In animals, cells undergoing apoptosis and necrosis are rapidly internalized by other cells via phagocytosis (engulfment) and degraded inside phagocytes. The removal of these dying cells provides a safe means for eliminating unwanted and dangerous cells from the body and actively modulates immune responses. The study of apoptotic cell removal will shed light on inflammatory and auto-immune disorders, many of which are associated with an inefficient clearance of apoptotic cells from the human body. This study is also closely related to cancer research and treatment. My long-term objective is to understand the molecular mechanism that controls the recognition, engulfment, and degradation of apoptotic and necrotic cells. I use the nematode Caenorhabditis elegans, a small round worm as a model organism to identify genes and delineate pathways that control these evolutionarily conserved events and will apply the knowledge to understand similar processes in mammals, including humans. We have identified novel signaling events both upstream and downstream of C. elegans phagocytic receptor CED-1, which have provided conceptual advances in our understanding of how apoptotic cells are removed by engulfing cells. In the next project period we propose to broaden our research scope by investigating the mechanisms behind these events. We have identified phosphatidylserine (PS) as one of the "eat me" signals exposed on the surface of apoptotic cells and activate CED-1. We will further study the molecular mechanisms for the exposure and recognition of PS and other potential "eat me" signals in both apoptotic and engulfing cells (Aim 1). We will expand our study to the recognition and phagocytosis of necrotic touch neurons, which die in a caspase-independent manner and may employ unique mechanisms to attract engulfing cells (Aim 1). We have discovered that phagocytic receptor CED-1 not only initiates the engulfment, but also promotes the degradation of apoptotic cells through the recruitment and activation of downstream mediator DYN-1, the C. elegans ortholog of mammalian large GTPase dynamin. We will study the mechanism leading to the transient enrichment of DYN-1 to the surface of extending pseudopodia and phagosomes, which is important for its functions in both engulfment and phagosome maturation. One particular hypothesis to test is that the CED-1 pathway recruits DYN-1 via the collaborative efforts of CED-6 and phosphatidylinositol 4,5- phosphate (PI(4,5)P2) (Aim 2). We have established C. elegans as a unique model system for studying the degradation of apoptotic cells inside phagosomes, a process not well studied, and have identified the specific functions of two Rab GTPases, a PI3 kinase and its product phosphatidylinositol 3-phosphate (PI(3)P) in phagosome maturation. To reveal the mechanism of PI(3)P function and the relationship between PI(3)P and Rab GTPases, we will identify downstream effectors of PI(3)P and the events that they regulate (Aim 3). PUBLIC HEALTH RELEVANCE: This project studies how unwanted cells (cells that commit suicide or die due to injuries) are recognized, internalized, and digested by their engulfing cells, a process that protects human bodies from their harmful effects. Understanding the mechanisms controlling this process will have important therapeutic implications, since many inflammatory and auto-immune diseases are closely related to defects in removing dying cells from human bodies, and will further help develop new strategies to specifically eliminate tumor cells. This project will be conducted in a small round worm the nematode C. elegans, which uses evolutionarily conserved mechanisms to control cell death, and provides a powerful means to reveal the principle for biological actions in a relatively simple system.